Scanning probe microscopy (SPM) is a measurement and analysis technique in which a measuring probe scans a sample of a measurement medium to be examined and a topography of the sample is determined via a distance-dependent interaction between the measuring probe and the sample. However, matter constants or other sample information can also be obtained. The most prominent examples of this technique are the atomic force microscope (AFM) and the scanning tunneling microscope (STM). Further examples of this technology are in particular the scanning near field microscope (SNOM) and the scanning photon microscope (SPhM). Furthermore, an examination method classified as scanning probe microscopy is the recording of a force-distance curve in which the measuring probe is essentially only displaced along a vertical direction in relation to the examined sample for measurement purposes.
To measure the distance-dependent interaction between measuring probe and sample, when performing a distance spectroscopy, the measuring probe is displaced in relation to the surface, for example in a direction vertical to the sample surface, and the interaction between measuring probe and sample is measured. Alternatively, the sample can also be moved. Furthermore, a relative movement between measuring probe and sample can be provided for in which both the measuring probe and the sample are moved. When performing a scanning probe microscopy, for example, this distance spectroscopy to measure the interaction between measuring probe and sample can be used to measure forces between molecules by binding a molecule to the measuring probe and another molecule to the sample. Thereafter, the interaction between the two bound molecules can be measured. However, intra-molecular forces can also be measured by e.g. lowering the measuring probe onto the sample and in the course of this, waiting for them to bond. The measuring probe can then be removed again from the sample, the forces acting on the measuring probe being recorded in the course of this. Additionally, further measurements are possible in which an interaction is measured which correlates with two or more points with an allocated distance.
In scanning force microscopy, a component which is also referred to as a cantilever is usually used as the measuring probe. This enables to measure forces by detecting the deformation of the measuring probe. To minimize the degree of interaction and thus to improve the lateral resolution, in most cases, a measurement tip is attached to the free end of the cantilever. The following explanations refer to a cantilever without limiting the generality. The explanations accordingly apply to other forms of measuring probes in probe microscopy. The cantilevers are typically fixed to a base, in particular to ensure appropriate handling.
It is known that both untreated and pre-treated cantilevers are used as measuring probes in distance spectroscopy. In the case of an untreated cantilever, binding of the sample during the measurement is non-specific. For example, in this connection, this refers to drawing molecules from their ambient medium by binding to the cantilever to measure the interaction of the molecules with the ambient medium. However, the molecules which are drawn can also be characterized more precisely in this connection. DNA molecules, for examples, display a specific spectroscopy curve due to an internal conformation transformation.
Especially specific bonds can be examined with a pre-treated cantilever. Such an examination can be advantageous if the formation of undesired bonds which thereafter can possibly hardly be separated from each other again is to be prevented during the measurement. It is therefore usual practice to bind one or more molecules to the measuring probe implemented as a cantilever which then forms a receptor-ligand system with the bound molecule(s). It is also known to bind whole cells to a measuring probe forming the cantilever and cause an interaction of this system with a sample, for example a biological material, or with other cells. In this case, it can be advantageous, for example, to use a cantilever without a tip. Pre-treatments of measuring probes, in particular of cantilevers, are known in different embodiments, for example in the form of hydrophobizing the measuring probe.
Known possibilities to pre-treat the cantilever generally lead to the coating of the measuring probe, in least in sections. A cell attached to the cantilever thus covers a section of the surface of the cantilever. In this connection, it can be planned to initially provide the cantilever with a coating in the course of the pre-treatment, in particular an adhesion-promoting coating onto which subsequently a substance to be measured is applied. Generally, in the following, the material applied to the measuring probe, in particular the cantilever, in the course of the pre-treatment is referred to as a probe substance, it being a single material or a combination of several materials which comprises an adhesion-promoting base and a substance arranged on this and to be examined, for example. A (base) coating being applied in the course of the pre-treatment and being comprised by the probe substance is also referred to as a probe coating.
If a pre-treated cantilever is used in the distance spectroscopy, several handling problems result in practice. The probe substance applied to the measuring probe is usually burdened by each mechanical contact with the sample in such a way that ageing of the probe substance occurs after one or more individual experiments. In the present application, the term “ageing” is very generally used for a change in the desired state of the probe substance which was brought about at the start of the distance spectroscopy measurements for the purposes of the measurement. However, ageing can not only occur due to the implementation of measurements, but also without such a measurement, for example, when using a probe substance in the form of a cell, no physiological conditions set for the cell are given before or also during the measurement.
The ageing of the probe substance can also result in decreasingly less specific bonds taking place during the implementation of the distance spectroscopy measurement, whereby the number of individual experiments has to be increased significantly. In a less specifically performed individual measurement, particular attention has to be paid to the fact that the measuring probe still contains the probe substance in the required manner in each experiment. Thus, for example, it would be possible for a probe substance implemented as a hydrophobic coating to slowly convert into a hydrophilic coating which would bias the measurement results severely. The implementation of the probe substance as a cell coating in which often only one cell fits onto the cantilever can result in the cell dying and changing very significantly already before performing the measurement experiment, thus it not being possible any longer to perform the actual measurement. This can also lead to incorrect measurement results if this event is taking place unnoticed. Using an untreated cantilever can lead to an adsorbate being formed through the contact with the sample such that even unspecific bonds are no longer possible.
Besides the measurements mentioned above, other measurements are also affected by the damaging, for example imaging methods, in particular by the damaging of the probe tip relevant to the interactions. If a surface topography is determined, for example, this is in particular influenced by the geometry of the tip of the measuring probe. Lithography shall be mentioned as another important example in which, for example, a structure is scratched into a sample by means of a tip and this structure is subsequently to be imaged once more for control purposes. However, as the tip is damaged by the scratching process, it is no longer possible to obtain an image of good quality.
If an ageing process interfering with or even preventing the measurement or else another undesired damaging of the measuring probe has occurred, this being pre-treated or untreated, the measuring probe is usually replaced. This replacement of the measuring probe takes time as it normally can only be performed manually. If the measuring probe is a cantilever, for example, this is fixed to a base which also serves to permit to position the cantilever without any destruction. This base is typically integrated into a measuring probe receptacle which is held in the scanning probe microscope. The different implementations of this include, for example, supporting it with a spring or supporting it by means of vacuum. This support now has to be detached when replacing the measuring probe to permit to insert a new measuring probe.
Another disadvantage of the replacement of the probe is that a contamination of the measurement environment, for example the fluid or also the sample itself, becomes more likely with each replacement process.
Furthermore, a disadvantage is that after the replacement of the base, it is no longer possible to find a sample point again at once. A solution for this exists, for example, in that the measuring probe base is structured and this structure is positioned in a precise distance to the measuring probe tip. If now a corresponding measuring probe receptacle is present in the scanning probe microscope, finding the structure again with the measuring probe tip is made possible. However, this procedure involves considerable expenditure both in terms of the production of the measuring probe and the equipment.